Abstract：Objective: To evaluate the potential protective effect of wogonin on hypoxic pulmonary hypertension (HPH) and to elucidate the molecular mechanism underlying these advantageous outcomes.Methods: In vivo,18 C57BL/6 male wild-type mice were randomly divided as normoxia group (N), hypoxiagroup (H+Saline) and hypoxia+wogonin group (H+w). The mouse hemodynamics were detected using a pressuresensor system 21 days after modeling. The degree of pulmonary artery remodeling was calculated by wall area/total area and wall thickness/total thickness. Prediction and screening of potential targets and molecular signalingpathways of wogonin in hypoxic pulmonary hypertension were performed based on network pharmacology. In vitro, mouse pulmonary artery smooth muscle cells (MPASMCs) were cultured under the respective condition of normoxia (N), hypoxia (H+PBS), hypoxia+low dose wogonin (H+40 μmol/L w), hypoxia+high dose wogonin (H+80 μmol/L w), hypoxia+high dose wogonin (H+80 μmol/L w)+740Y-P (20 μg/mL). The N group was exposed to normoxic (5% CO2, 21% O2, 74% N2, 37 ℃) environment for 48 h. The H+PBS, H+40 μmol/L w, H+80 μmol/L w and H+80 μmol/L w+740Y-P groups were exposed to hypoxic (5% CO2, 3% O2, 92% N2, 37 ℃) environment for 48 h. Cell proliferation was assessed with the CCK-8 and EdU assay, respectively. The migration capacity of MPASMCs was determined by transwell and scratch wound healing assays. Protein expression profiles of the PI3K/AKT/RXRA/Bcl-2 signaling pathway (P-PI3K, PI3K, P-AKT, AKT, RXRA, Bcl-2) were analyzed using the Western blot method and the PI3K pathway activator 740Y-P was used for functional complementation experiments. The expression level of miR-451 was detected using RT-qPCR to assess the impact of wogonin.The expression of CAB39 and MIF, upstream regulatory factors of the PI3K/AKT/RXRA/Bcl-2 pathway, was examined by Western blot to detect the effect of wogonin on their regulation. Results: Comparative to the controls,hypoxic MPASMCs showed significantly increased proliferation and migration abilities (P<0.05). Moreover,the hypoxia group exhibited a significant increase in right ventricular systolic pressure (RVSP) and pulmonary vascular remodeling in comparison to the control group (P<0.05). After intervention with wogonin, the proliferation and migration activity of MPASMCs, RVSP and pulmonary vascular remodeling were rescued (P<0.05). The hypoxia group exhibited a significant increase in the expressions of p-PI3K, p-AKT, RXRA, and Bcl-2, which are proteins related to the PI3K/AKT/RXRA/Bcl-2 signaling pathway, when compared to the normoxia group.However, after wogonin intervention, the PI3K/AKT/RXRA/Bcl-2 signaling pathway was significantly inhibited(P<0.05). Functional restoration experiments confirmed that under hypoxic conditions, activation of the PI3K pathway can significantly weaken the inhibitory effect of wogonin on the proliferation of MPASMCs. In the context of the PI3K/AKT/RXRA/Bcl-2 pathway, it has been observed that wogonin exhibited a notable inhibitory effect on the upstream regulators CAB39 and MIF expression. Compared with to the normoxia group, the expression levels of CAB39 and MIF exhibited a significant up-regulation, while the expression of miR-451 demonstrated a significant downregulation in the hypoxia group (P<0.05). After wogonin intervention, the expression levels of CAB39, MIF and miR-451 were reversed. Further experimentation suggested that the intervention of a miR- 451 inhibitor demonstrated a partial reversal of the inhibitory impact exerted by wogonin on the proliferation and migration of MPASMCs (P<0.05). Conclusion: Wogonin has the ability to hinder the PI3K/AKT/RXRA/Bcl-2 signaling pathway through the up-regulation of miR-451 expression. This intervention leads to the amelioration of abnormal proliferation and migration in hypoxic MPASMCs, as well as the remodeling of pulmonary vascular structure in mice afflicted with HPH. Consequently, the administration of wogonin results in a reduction of pulmonary hypertension.
